In our initial studies of the regulation of RORgamma-t transcription we showed with luciferase reporter constructs driven by RORgamma-t promoter fragments of various lengths that mutation of particular E-protein binding sites (E-Boxes) led to reduced promoter-reporter activity. In addition, we showed that mutation of a Runx1 consensus binding site also caused reduced promoter-reporter activity. In further studies in which we evaluated the function of E-proteins in physiological CD4+ T cells stimulated under Th17 conditions we found that E-proteins bind to the RORgamma-t promoter. In addition, transfection of CD4+ T cells with E-protein-specific siRNA led to reduced RORgamma-t expression. The above studies suggested that E-proteins regulate RORgamma-t expression. To directly investigate this possibility we performed extensive studies of conditional E-protein KO mice which have phloxed E-protein genes and a T cell-specific Cre transgene under a tamoxifen responsive promoter. Thus, when cells from the mice were exposed to tamoxifen E-protein expression is inhibited. Using a number of different tamoxifen administration regimens we achieved as much as 75-80% deletion of E-proteins and found that in such deletion was associated with a comparable deletion in RORgamma-t expression and IL-17 expression. Thus, is is clear that E-proteins are important regulators of ROR-gamma-t expression. In related studies we investigated the role of Id proteins in RORgamma-t expression. This was likely since Id proteins bind to E-proteins and modify their function. In expectedly, we found that mice with complete Id3-deficiency (and to a lesser extent Id2 deficiency) have reduced RORgamma-t expression. This proved to be due to the fact that Id3 inhibits IL-4 expression and IL-4 induction of GATA-3, the latter an inhibitor of RORgamma-t expression. Thus, Id3 has a net positive effect on RORgamma-t expression because it suppresses the appearance of an RORgamma-t inhibitor. In a related set of studies we found that E-protein levels were greatly enhanced by a combination of TGF-beta and IL-6, the two cytokines that are necessary for induction of the Th17 response. Thus it seems likely that a major function of these inducing cytokines in establishing the Th17 response is to induce E-proteins. Finally, we tested the importance of E-proteins to the Th17 response with studies of experimental allergic encephalomyelitis(EAE). Here we showed that in two different models of EAE, mice with T cells deficient in E-proteins manifest greatly attenuated EAE. These in vivo studies thus confirmed the conclusion from the above in vitro studies establishing E-proteins as major regulators of the Th17 response.
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